This application is for competitive renewal of a highly productive R01 research program focused on elucidating the roles of microRNAs (miRNAs) in key signaling pathways that drive tumorigenesis. miRNAs are 18-24 nucleotide RNA molecules that regulate the stability and translational efficiency of partially complementary target messenger RNAs. Over the last decade, many laboratories including ours have established that miRNAs are frequently dysregulated in cancer cells and can influence all aspects of malignancy including increased proliferation, resistance to apoptosis, and metastasis. Moreover, our work funded by this grant has documented that miRNAs provide crucial functions downstream of classic oncogenes and tumor suppressors including MYC, KRAS, and p53. These findings led to our demonstration that miRNA-based therapeutic strategies can potently inhibit tumorigenesis in animal models. Nevertheless, many important questions remain unanswered. We still do not fully understand the mechanisms that result in abnormal miRNA expression in tumors. Furthermore, we know very little about the molecular mechanisms through which gain- and loss-of-function of miRNAs drives tumorigenesis in vivo. The vast majority of existing functional data has been derived from altering miRNA expression in cell lines, an approach that does not fully model how miRNAs participate in tumor initiation and progression. During the next funding period of this grant, we propose to address these critical knowledge gaps by testing the following central hypotheses: First, that alternative splicing of miRNA primary transcripts and association of sequence-specific RNA binding proteins influences the expression and activity of oncogenic and tumor suppressor miRNAs; and second, that the activity of anti-tumorigenic miRNAs can suppress early- and late-stage liver tumorigenesis and lymphomagenesis in vivo. Three Specific Aims will be pursued in order to test these hypotheses. In Aim 1, we will use cellular and animal models to directly determine the role of alternative splicing in regulating miRNA production and activity in developmental and disease contexts, including tumor models. In Aim 2, the role of specific RNA binding proteins in regulating the biogenesis of a set of anti-tumorigenic miRNAs will be investigated. Finally, in Aim 3, we will utilize a newly-developed mouse model with regulatable miRNA expression to investigate the mechanisms underlying miRNA-mediated tumor suppression in mouse models of liver cancer and lymphoma. These aims will take advantage of our extensive experience, and that of our collaborators, in evaluating miRNA regulation and function in vitro and in vivo. We anticipate that the principles revealed by these studies will be broadly applicable to our understanding of the roles of miRNAs in cancer and may uncover new opportunities to manipulate miRNA expression and function for therapeutic purposes.